基于CRISPR-Cas9基因编辑技术的短程硝化颗粒污泥去除药品类新兴污染物的机理研究

Pharmaceutical emerging contaminants (ECs) were detected frequently in municipal wastewater treatment plants and nature environment. It is extremely urgent to eliminate pharmaceutical ECs effectively. Nitrifying activated sludge has better removal performance for some pharmaceutical ECs compared with conventional activated sludge. However the removal mechanism of pharmaceutical ECs by nitrifying sludge is still not clear. The effect of nitrifying sludge granulation on pharmaceutical ECs removal should be studied. CRISPR-Cas9 gene editing technique has been seen as a breakthrough finding by Science in 2015 due to its amazing virtues of simple and high throughput. However, the CRISPR-Cas9 technique has not been used in the study of environment field. In this study, the ammonia oxidation functional genes and AHL quorum sensing functional genes of pure ammonia oxidizing bacteria (AOB) and nitrifying sludge will be knocked out selectively by using CRISPR-Cas9 gene editing technique. The effect of ammonia oxidation functional genes and quorum sensing system on pharmaceutical ECs removal will be investigated. The mechanism and key factors of the co-metabolism between partial nitrification and pharmaceutical ECs degradation by AOB will be illuminated. Whether the granulation of the nitrifying sludge can enhance the expression of functional genes mentioned above and pharmaceutical ECs removal will be verified. The relationship among functional genes, nitrifying sludge granulation and pharmaceutical ECs removal will be defined. The results of our study could contribute to optimize the configuration of biological wastewater treatment plants to improve removal of some of the more persistent pharmaceuticals and other ECs in wastewater before they are discharged into the environment.

药品类新兴污染物（ECs）在污水处理系统及自然界中频繁检出，如何有效去除迫在眉睫。研究表明硝化污泥能去除药品类ECs，但其去除机理以及硝化污泥颗粒化的作用有待研究。作为《科学》2015年“突破性发现”的CRISPR-Cas9基因编辑技术具有通量高、成本低等特点，尚未有环境领域相关研究报道。本申请拟运用CRISPR-Cas9技术，对氨氧化菌（纯种）以及硝化污泥（混合菌群）中的功能基因(氨氧化和群体感应）进行选择性敲除，考察上述功能基因表达对药品类ECs去除（吸附和降解）的影响，阐明氨氧化菌对药品类ECs共代谢的机理及关键因素，考察短程硝化污泥颗粒化对上述功能基因表达和药品类ECs去除的影响，明确功能基因（氨氧化和群体感应）、污泥颗粒化以及药品类ECs去除之间的内在关系。项目成果将为强化生物处理工艺同步硝化和去除药品类ECs提供方法，并能为研究自然界中氮循环与药品类ECs共代谢提供理论依据。

硝化污泥被报道能去除药品类新兴污染物（ECs），但其去除机理及硝化污泥颗粒化的作用未知。本项目研究表明自养好氧氨氧化菌N.europaea去除17α-乙炔雌二醇(EE2)包括生物共代谢和非生物硝基化两种途径，氨氮是EE2共代谢发生的必要条件，当pH大于7.5时，N.europaea对EE2的去除以生物降解为主，生物降解反应符合一级动力学模型且降解速率常数为0.0069h-1，当pH低于7.5时，在生物降解和非生物硝基化的协同作用下，EE2去除符合一级动力学模型且降解速率常数为0.0093h-1，EE2的非生物转化是由氨氧化细菌生成的FNA介导而不是亚硝氮，EE2初始浓度和pH也会影响EE2的转化效率，FNA积累产生的NO2是EE2在酸性条件下被化学转化的关键活性物质，而作为氨氧化中间体的羟胺对EE2无转化能力，NO仅在碱性条件下对EE2有转化作用，EE2被硝基化后可有效降低其雌激素效应且反应产物不发生逆反应。此外，通过构建和电导入携带Cas9蛋白编码基因的广宿主质粒，从转录和表达两个角度验证了外源Cas9蛋白能在N.europaea体内合成，通过对编辑后的目标功能基因测序分析，证明了应用Crispr-Cas9技术编辑N.europaea功能基因的可行性。.研究表明硝化活性污泥（NAS）颗粒化对EE2去除具有积极影响，硝化颗粒污泥（NGS）对EE2的去除效率为3.705μgEE2/(gMLSS·h)，明显优于NAS（1.695μgEE2/(gMLSS•h)），通过人工神经网络模型和Spearman相关性分析表明，NGS中的亚硝酸盐积累是影响EE2去除的关键因素，在EE2的胁迫下NGS氮循环amoA和nirK基因丰度下降但表达上调，NGS中高浓度胞外蛋白和群体感应信号分子有助于吸附降解EE2，在异养菌与自养菌协同去除EE2方面具有明显优势；经实验研究和文献调研分析，药品类ECs在脱氮工艺中的降解机理包括自养氨氧化菌生物转化、非生物转化及逆反应、异养微生物降解和共代谢/非生物转化协同异养微生物矿化，而药品类ECs中间代谢产物的生物毒性因种类和结构而异。.本项目系统分析了影响药品类ECs在短程硝化工艺中去除效率的关键因素，从生物及化学角度阐述了自养菌、异养菌及活性氮物质在药品类ECs去除过程中的作用，为强化生物处理工艺同步硝化和去除药品类ECs提供理论依据和调控方法。